PROJECT ABSTRACT Recent scientific advancements have made immunotherapy a promising option for many cancer patients. Immunotherapy can bolster the ability of the immune system to recognize and destroy cancer cells. However, not all patients respond to immunotherapy, thus a better understanding of the mechanisms that underlie immune cell effector function is required to improve response rates. In collaboration with Dr. Carl June's group, our lab recently studied a CLL patient who had gone into complete remission following CAR-T therapy. We found that an overwhelming majority of the CAR+ cells had a disruption in the TET2 gene, and experimental knockdown of TET2 resulted in enhanced proliferative capacity and anti-tumor activity of CAR T cells in vitro. Furthermore, T cells with the TET2 knockdown displayed increased expression of TOX2, a member of the HMG-box family of DNA-binding proteins. TOX2 is an immune-specific transcription factor expressed in the human spleen and tonsils. Interestingly, the only known function of TOX2 is that it positively regulates the transcription factor T-BET during the development of natural killer cells, by repressing the inhibitory PD-1 protein. Owing to the increase in TOX2 mRNA levels in TET2 knockdown CAR T cells, in addition to its ability to downregulate a key inhibitory receptor, I hypothesize that TOX2 is a positive regulator of T cell effector function due to its ability to upregulate T-BET. To test this hypothesis, I will pursue three aims. Aim 1 is to manipulate TOX2 expression in human T cells to determine whether it improves T cell differentiation and effector function. Aim 2 is to determine the pattern of TOX2 binding to the genome, with the goal of identifying its transcriptional targets, at both the mRNA and protein levels. Very little is known about how HMG-box proteins bind chromatin in vivo. Thus, this aim will also provide a more fundamental understanding of the chromatin functions of HMG proteins. Aim 3 is to determine levels of TOX2 mRNA in CLL patients who have received CAR T therapy, to determine whether higher TOX2 levels are predictive of better response to CAR T therapy. This study could elevate TOX2 as a novel target for improving CAR T cell function. An understanding of the molecular mechanisms could make it possible to activate TOX2, ultimately improving patient responses to immunotherapy.